Excision of transgenes in genetically modified organisms

ABSTRACT

A method for deleting a region of DNA in a plant. In some embodiments, the method comprises transforming a plant with a nucleic acid molecule, wherein the nucleic acid molecule encodes one or more zinc finger nuclease(s) (ZFNs) operably linked to one or more tissue-specific promoter(s), e.g., a pollen-specific promoter. Methods include excising native genes in a plant. Accordingly, in some embodiments, ZFNs are engineered that recognize sequences that flank native plant genes. In further embodiments, ZFNs are expressed under the control of developmental stage-specific promoters, such that, for example, nucleic acid sequences are specifically excised in plants during relatively late stages of development. Nucleic acid molecules useful for carrying out disclosed methods and plants produced by the methods are included.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/011,666 filed Jan. 21, 2011 which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/297,628, filed Jan. 22, 2010,the disclosure of each are hereby incorporated herein in their entiretyby this reference.

FIELD OF THE INVENTION

The invention generally relates to compositions and methods forgenerating transgenic plants. In certain embodiments, the transgenicplants comprise one or more transgenes of interest. In certainembodiments, excision of transgene(s) is directed in pollen and/or seed,such that the pollen and/or seed produced by a transgenic plant of theinvention is substantially free of transgene(s). In some embodiments,transgenic plants of the invention are useful, for example, in achievingbioconfinement of transgene(s) of interest in the transgenic plant. Inother embodiments, the excision of the transgene is directed to aspecific expression cassette, such as a selectable marker, such thatonly this expression cassette is removed from the transgenic plantand/or progeny of the transgenic plant.

BACKGROUND

Many plants are genetically transformed with genes from other species tointroduce desirable traits, such as to improve agricultural valuethrough, e.g., improving nutritional value quality, increasing yield,conferring pest or disease resistance, increasing drought and stresstolerance, improving horticultural qualities such as pigmentation andgrowth, and/or imparting herbicide resistance; enabling the productionof industrially useful compounds and/or materials from the plant; and/orenabling the production of pharmaceuticals. The introduction of clonedgenes into plant cells and recovery of stable fertile transgenic plantscan be used to make such modifications of a plant, and has alloweddesirable traits or qualities of interest to be incorporated into plantsvia genetic engineering (e.g., crop improvement). In these methods,foreign DNA is typically introduced into the nuclear or plastid DNA ofthe eukaryotic plant cell, followed by isolation of cells containing theforeign DNA integrated into the cell's DNA, to produce stablytransformed plant cells.

One drawback that arises regarding the use of transgenic plants is thepossibility of transgene escape to wild species and non-transformedspecies. These traits can increase the risk of outcrossing, persistence,and introgression of transgenes into an adjacent population. The escapeof transgenes from genetically modified (GM) crops usually occursthrough gene flow, mainly by cross-pollination (Lu (2003) Eviron.Biosafety Res. 2:3-8), but may also occur through introgression. StewartJr. et al. (2003) Nat. Reviews Gen. 4:806-17. Crop-to-crop gene flowwill result in contamination of non-GM varieties, affecting thestrategic deployment of transgenic and non-transgenic crop varieties ina given agricultural system. Significant contamination of non-GM cropswith transgenic material poses difficulties in international tradebecause of legal restrictions on imports of transgenic products by manycountries. Crop-to-crop gene flow can cause stacking of transgenes inhybrids that may potentially become volunteer weeds if the transgenesimpart multiple resistance (e.g., to herbicides, pests, and/ordiseases). Additionally, crop-to-crop gene flow will lead to transgeneescape into weedy populations or related wild species, which may poseserious weed problems and other ecological risks if the transgenespersist and establish in the weedy/wild populations through sexualreproduction and/or vegetative propagation. This is a particular concernwhen escaped genes enhance the ecological fitness of the weedy/wildspecies. Introgression of a crop transgene occurs in steps involvingseveral successive hybrid generations. Introgression is a dynamicprocess that may take many years and generations before the transgene isfixed in the genetic background of a receiving species and, thus,presents difficulties of detection and monitoring. However, if selectionis strong and/or population size is small, fixation of an introgressedgene may occur rapidly.

Containment of a specific expression cassette within geneticallymodified plants, especially a selectable marker expression cassette, isan elusive goal. Selectable marker genes are usually antibioticresistant or herbicide tolerant genes, but may include reporter genes(i.e., ß-glucuronidase (Graham et al. (1989) Plant Cell Tiss. Org.20(1):35-39). Selectable makers which are co-transferred into the genomeof a plant provide a selective advantage and allow for theidentification of stably transformed transgenic plants. The availabilityof functional selectable maker genes which can be used for thetransformation of plants is somewhat limited. A review of the publishedscientific literature on transgenic crop plants reveals that the mostwidely used selective agents for antibiotic resistance are for kanamycin(encoded by the neomycin phosphotransferase type II gene (Bevan et al.(1983) Nature 304:184-187)) or hygromycin (encoded by the hygromycinphosphotransferase gene (Waldron et al., Plant Mol. Biol. 5:103-108)),and herbicide tolerance is phosphinothricin resistance (encoded by thepat (Wohlleben et al. (1988) Gene 70:25-37) or bar genes (DeBlock et al.(1987), EMBO J. 6 (9):2513-2518)). See, Sundar et al. (2008) J. PlantPhysiol. 165:1698-1716. Given the limited number of selectable markergenes and the common use of a sub-set of these traits, a solution thatallows for the excision and re-use of selectable markers within atransgenic plant would obviate the need for additional selectable makersin subsequent rounds of gene transfer or gene stacking into the sameplant. Moreover, the ability to excise a selectable marker couldovercome unintended changes to the plant transcriptome that are causedby the expression of the marker (Abdeen et al. (2009) Plant Biotechnol.J. 7(3):211-218).

Current strategies to prevent or minimize gene flow between GM crops andother species and varieties include: (1) physical isolation of thetransgenic crop; (2) chloroplast engineering of transgenes; (3)co-engineering of a mitigation gene along with the transgene; (4)genetic use restriction technologies (GURTs); (5) CRE/loxP and FLP/FRTrecombinase-mediated gene deletion. See, e.g., Lee and Natesan (2006)TRENDS Biotech. 24(3):109-14; Lu (2003), supra; and Luo et al. (2007),Plant Biotech. J. 5:263-74; and (6) meganuclease—mediated gene deletion.See, e.g., U.S. patent application Ser. No. 11/910,515; and U.S. patentapplication Ser. No. 12/600,902.

CRE, FLP, and R recombinases have been exploited for the excision ofunwanted genetic material from plants. Hare and Chua (2002) Nat.Biotech. 20:575-80. Luo et al. (2007), supra, reported a pollen- andseed-specific “GM-gene-deletor” system, wherein use of loxP-FRT fusionsequences as recognition sites for excision of transgenes by CRE or FLPrecombinase led to deletion of transgenes from pollen, or from bothpollen and seed, of transgenic tobacco plants. All these site-specificrecombinase systems shown to function in plants are members of theintegrase family. These systems have been chosen for use, at least inpart, due to the fact that other recombinases may require ancillaryproteins and more complex recognition sites that may confer topologicalrestraints on recombination efficiencies. Id. These systems have severalsignificant drawbacks: integrase-type recombinases may also recognize“pseudo-sequences,” which may be highly divergent from a specific targetsequence and, therefore, lead to unwanted non-specific DNA deletions;and excision of a target sequence leaves a residual recognition sequencethat may be sites of chromosomal rearrangements upon subsequent exposureto the recombinase, or activate gene silencing mechanisms. Id. Moreover,these systems are further constrained as a functional recombinase mustbe present and expressed in one of the parent plants, the presence ofwhich requires additional strategies for deletion within pollen and/orseed. Despite these limitations, the CRE/loxP system is recognized asthe most suitable strategy for optimization of gene deletion in plants.Id.

Custom-designed zinc finger nucleases (ZFNs) are proteins designed todeliver a targeted site-specific double-strand break in DNA, withsubsequent recombination of the cleaved ends. ZFNs combine thenon-specific cleavage domain of FokI restriction endonuclease with zincfinger DNA-binding proteins. See, e.g., Huang et al. (1996) J. ProteinChem. 15:481-9; Kim et al. (1997) Proc. Natl. Acad. Sci. USA 94:3616-20;Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93:1156-60; Kim et al.(1994) Proc. Natl. Acad. Sci. USA 91:883-7; Kim et al. (1997b) Proc.Natl. Acad. Sci. USA 94:12875-9; Kim et al. (1997c) Gene 203:43-9; Kimet al. (1998) Biol. Chem. 379:489-95; Nahon and Raveh (1998) NucleicAcids Res. 26:1233-9; Smith et al. (1999) Nucleic Acids Res. 27:674-81.Individual zinc finger motifs can be designed to target and bind to alarge range of DNA sites. Cys₂His₂ zinc finger proteins bind DNA byinserting an α-helix into the major groove of the double helix.Recognition of DNA by zinc fingers is modular: each finger contactsprimarily three consecutive base pairs in the target, and a few keyresidues in the protein mediate recognition. It has been shown that FokIrestriction endonuclease must dimerize via the nuclease domain in orderto cleave DNA, inducing a double-strand break. Similarly, ZFNs alsorequire dimerization of the nuclease domain in order to cut DNA. Mani etal. (2005) Biochem. Biophys. Res. Commun. 334:1191-7; Smith et al.(2000) Nucleic Acids Res. 28:3361-9. Dimerization of the ZFN isfacilitated by two adjacent, oppositely oriented binding sites. Id. Inaddition, double strand breaks caused by zinc finger nucleases areresolved by the plants DNA repair machinery via either nonhomologous endjoining (NHEJ) or homology directed repair (HDR), thereby resulting inplants which are free of residual recognition sequences.

SUMMARY OF THE DISCLOSURE

According to an embodiment of the invention, a method for deleting aregion of DNA in a plant wherein a viable plant containing a genomicDNA, the genomic DNA comprising the region of DNA, is provided; and azinc finger nuclease, engineered to cleave the genomic DNA at arecognition sequence, is expressed or introduced in the viable plantcontaining the genomic DNA; thereby resulting in cleavage of the genomicDNA at recognition sequences resulting in the excision of the genomicDNA, wherein the region of DNA is absent from the genomic DNA.

In another embodiment, a method for deleting a region of DNA in a plantincludes providing a first viable plant containing a genomic DNA, thegenomic DNA comprising the region of DNA and a first recognitionsequence flanking the 3′ end and a second recognition sequence flankingthe 5′ end of the region of DNA. A second viable plant containing agenomic DNA is provided, the genomic DNA comprising a DNA encoding azinc finger nuclease engineered to cleave the genomic DNA at therecognition sequences. The first and second viable plants are crossedsuch that F₁ seed is produced on either the first or the second viableplant. A resultant F₁ plant containing a genomic DNA is grown, whereinthe region of DNA is absent from the genomic DNA. In certainembodiments, the first recognition sequence and the second recognitionsequence can be identical.

In a particular embodiment, an isolated nucleic acid molecule includes:a first nucleic acid sequence recognized by a zinc finger nuclease; agene of interest; and a second nucleic acid sequence recognized by azinc finger nuclease, wherein the gene of interest is flanked by thefirst and second nucleic acid sequences recognized by a zinc fingernuclease. In another embodiment, the first recognition sequence and thesecond recognition sequence can be flanked by homologous sequences. Inyet another embodiment, a method of producing a transgenic plantincludes transforming a plant cell or plant tissue with the isolatednucleic acid molecule and regenerating a whole plant.

In an additional embodiment, a method for reducing the transmission of agene of interest to other plants includes crossing the whole plant witha plant regenerated from a plant cell or tissue transformed with anisolated nucleic acid molecule comprising a pollen-specific promoteroperably linked to a zinc finger nuclease, wherein the gene of interestis specifically excised in pollen of the progeny resulting from thecross. The progeny resulting from the cross are cultivated. In suchembodiment, an isolated nucleic acid molecule includes a promoter and anucleic acid sequence encoding a zinc finger nuclease, wherein thepromoter is operably linked to the nucleic acid sequence encoding thezinc finger nuclease and the method of producing a transgenic plant thatincludes transforming a plant cell or plant tissue with the isolatednucleic acid molecule and regenerating a whole plant.

In an embodiment, a method for deleting a region of DNA in a plantcontaining a nucleic acid molecule including: a first nucleic acidsequence recognized by a zinc finger nuclease; a selectable marker geneexpression cassette; and a second nucleic acid sequence recognized by azinc finger nuclease, wherein the selectable marker is flanked by thefirst and second nucleic acid sequences recognized by a zinc fingernuclease. In another embodiment, the first recognition sequence and thesecond recognition sequence are flanked by homologous sequences.Additionally, a zinc finger nuclease, engineered to cleave the genomicDNA at a recognition sequence, is expressed or introduced in the viableplant cell; thereby resulting in cleavage of the genomic DNA atrecognition sequences resulting in the excision of the genomic DNA,wherein the selectable marker is absent from the genomic DNA.

In another embodiment, each half of the zinc finger nuclease monomer isexpressed separately and when paired in conjunction with one anotherform a functional complex. For example, a plant transcription unit whichexpresses one zinc finger nuclease monomer (consisting of a zinc fingerbinding motif operably linked to the FokI endonuclease) is stablyintegrated into one parent, P1, and a plant transcription unit whichexpresses a second monomer is stably integrated into a second parent,P2. The sexual cross of P1×P2 results in progeny plants which containboth zinc finger monomers. The resulting zinc finger nuclease dimer iscapable of binding to a zinc finger binding site and forming a complexwhich has cleavage activity. Given that the Fold endonuclease is activeas a dimer (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95:10,570-10,575), the cleavage activity is only capable of occurringwithin progeny which contain both functionally expressing monomers.

In another embodiment, the excision by a zinc finger nuclease at arecognition sequence results in the formation of a cleavage junction,which is free of a residual recognition sequence. The cleavage junctionmay not be bound and cleaved by the original zinc finger nuclease(s).Additionally, the cleavage junction can be the result of non-homologousend joining (NHEJ) or the result of homology directed repair between twohomologous regions of DNA which are located upstream of the 5′recognition sequence and downstream of the 3′ recognition sequence orthe result of another undescribed DNA repair mechanism. A homologoussequence can be placed outside binding sites so that after cleavage,homology directed repair can occur. This is an improvement overrecombinase systems, which always leave behind a remnant of the siteused to get the excision.

In yet another embodiment, a method of excising a native gene ofinterest in a plant includes transforming a plant cell or tissuecomprising a gene of interest with an isolated nucleic acid moleculecomprising a nucleic acid sequence encoding a zinc finger nuclease or anisolated protein sequence which encodes a zinc finger nuclease, whereinthe zinc finger nuclease recognizes a nucleic acid sequence flanking thenative gene of interest and the native gene of interest is specificallyexcised. A whole plant is then regenerated. In an alternativeembodiment, endogenous gene excision can be accomplished by crossing aplant expressing a zinc finger nuclease with a target plant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 includes the plasmid map for plasmid pDAS5380.

FIG. 2 includes the plasmid map for plasmid pDAS5381.

FIG. 3a is a schematic diagram and restriction map of the T-DNA insert.FIG. 3b includes several panels depicting T₀ Southern blot analysis usedto identify events which contained full length intact PTUs from plasmidpDAS5380 according to an embodiment of the invention. The T₀ Southernblot analysis image used restriction enzymes MfeI and NsiI to digest thepDAS5380 events, showing intact T-DNA inserts by co-hybridization of GUSand PAT.

FIG. 4A is a schematic diagram and restriction map of the T-DNA insert.FIG. 4b includes several panels depicting T₀ Southern blot analysis usedto identify events which contained full length intact PTUs from plasmidpDAS5381 according to an embodiment of the invention. The T₀ Southernblot analysis image used restriction enzymes MfeI and NsiI to digest thepDAS5381 events, showing intact T-DNA inserts by hybridization of HptII.

FIG. 5 includes Southern blot analysis of a select group of events thatare representative of a larger sample according to an embodiment of theinvention. These samples were selected to illustrate the excisedfragment (i.e., the lower molecular fragment), the non-excised fragment(i.e., the higher molecular weight fragment), and the chimeric eventswhich contained both the excised and non-excised fragments. In addition,controls of the wild-type genomic DNA and 100 pg of the pDAS5380 plasmidwere included. This data correlated with the GUS expression data. Eventsthat did not stain positive via histochemical staining for GUS did notcontain a full-length, intact GUS PTU expression cassette.

FIG. 6 includes the image of an agarose gel containing PCR amplifiedfragments of the genomic DNA samples used in the Southern blotexperiments according to an embodiment of the invention. These PCRamplicons illustrate the excised fragment (i.e., the lower molecularweight fragment), the non-excised fragment (i.e., the higher molecularweight fragment), and the chimeric events which contained both theexcised and non-excised fragments. In addition, controls of the wild T₀plants are included; the larger intact GUS PTU expression cassette wasamplified in these reactions. Negative controls where wild-type genomicDNA and no DNA (H₂O) were used for the PCR reactions are also included.This data correlated with the GUS expression data and the Southern blotdata.

FIGS. 7a and 7b include an alignment of sequence analysis of the 2.4 kbband showing deletion of the GUS expression cassette according to anembodiment of the invention. The bold sequence indicates the At ActinPromoter and MAR gene elements. CCR5 binding sites are identified withunderlining and italics. Although multiple amplicons were generated andsequenced per event, only one amplicon was aligned in the Figures.

FIG. 8 includes PCR analysis of F₂ progenies of “Intact” F₁ hybridsaccording to an embodiment of the invention.

FIG. 9 includes Southern analysis of F₂ progenies of “Intact” F₁ hybridsaccording to an embodiment of the invention.

FIG. 10 includes PCR analysis of F₂ progenies of “Excised” F₁ hybridsaccording to an embodiment of the invention.

FIG. 11 includes Southern analysis of F₂ progenies of “Excised” F₁hybrids according to an embodiment of the invention.

FIG. 12 includes PCR analysis of F₂ progenies of “Chimeric” F₁ hybridsaccording to an embodiment of the invention.

FIG. 13 includes Southern analysis of F₂ progenies of “Chimeric” F₁hybrids according to an embodiment of the invention.

SEQUENCE LISTING

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases. Onlyone strand of each nucleic acid sequence is shown, but the complementarystrand is understood as being included by any reference to the displayedstrand. In the accompanying sequence listing:

SEQ ID NO:1 shows a CCR5 ZFN binding site.

SEQ ID NO:2 shows a CCR5 Zinc Finger Nuclease gene sequence.

SEQ ID NO:3 shows a TQPATS primer.

SEQ ID NO:4 shows a TQPATA primer.

SEQ ID NO:5 shows a TQPATFQ primer.

SEQ ID NO:6 shows a TQPALS primer.

SEQ ID NO:7 shows a TQPALA primer.

SEQ ID NO:8 shows a TQPALFQ primer.

SEQ ID NO:9 shows a HPT2S primer.

SEQ ID NO:10 shows a HPT2A primer.

SEQ ID NO:11 shows a HPTFQ primer.

SEQ ID NO:12 shows a FokI_UPL_F primer.

SEQ ID NO:13 shows a FokI_UPL_R primer.

SEQ ID NO:14 shows a BY2ACT89S primer.

SEQ ID NO:15 shows a BY2ACT89A primer.

SEQ ID NO:16 shows a forward PCR primer for PTU PCR analysis.

SEQ ID NO:17 shows a reverse PCR primer for PTU PCR analysis.

SEQ ID NO:18 shows a BYACTFQ primer.

DETAILED DESCRIPTION

Disclosed herein is a method to excise genes from specific plant tissuein genetically modified organisms. In some embodiments, one or more ZFNs(zinc finger nuclease) are used to remove transgenes from specific planttissue as a means of reducing gene flow into non-GM crops. In someembodiments, the transgene that is removed is a selectable marker genecassette. In certain embodiments, the specific plant tissue is pollen.

In some embodiments, one or more ZFNs may be operably linked todifferent tissue-specific promoters. In these and further embodiments,one of the one or more ZFNs operably linked to a tissue-specificpromoter may be transformed into one parent plant line, and another ofthe one or more ZFNs operably linked to a different tissue-specificpromoter may be transformed into a second parent plant line. A crossbetween the parental lines containing each of the one or more ZFNs canproduce an F₁ line that contains a functional ZFN that cleaves DNA at arecognition sequence. The recognition sequences may flank transgenes inthe DNA of the plant.

Tissue-specific gene excision may be achieved by operable linkage oftissue-specific plant promoters to ZFNs. In some embodiments, operablelinkage of a tissue-specific promoter to one or more ZFNs leads totissue-specific expression of the one or more ZFNs, thereby excising theZFNs, selectable markers, and/or any genes or nucleic acid sequenceslocated between the recognition sequences in the specific tissue.

In particular embodiments, one or more ZFNs are expressed within thesame plant. ZFNs may be operably linked to promoters that driveexpression of the ZFNs during later developmental stages of a plant. Inthese and other embodiments, one or more functional ZFNs may cleavespecific recognition sequences that flank one or more transgene(s),thereby removing the one or more transgenes from plant tissue duringlater stages of plant development.

Abbreviations

GM Genetically modified

PTU Plant transcription unit

ZF Zinc finger

ZFN Zinc finger nuclease

ZFP Zinc finger protein

Terms

Gene expression: The process by which the coded information of a nucleicacid transcriptional unit (including, e.g., genomic DNA or cDNA) isconverted into an operational, non-operational, or structural part of acell, often including the synthesis of a protein. Gene expression can beinfluenced by external signals; for example, exposure of a cell, tissue,or organism to an agent that increases or decreases gene expression.Expression of a gene can also be regulated anywhere in the pathway fromDNA to RNA to protein. Regulation of gene expression occurs, forexample, through controls acting on transcription, translation, RNAtransport and processing, degradation of intermediary molecules such asmRNA, or through activation, inactivation, compartmentalization, ordegradation of specific protein molecules after they have been made, orby combinations thereof. Gene expression can be measured at the RNAlevel or the protein level by any method known in the art, including,without limitation, Northern blot, RT-PCR, Western blot, or in vitro, insitu, or in vivo protein activity assay(s).

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidmolecules consist of nitrogenous bases that are either pyrimidines(cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) andguanine (G)). These nitrogenous bases form hydrogen bonds between apyrimidine and a purine, and the bonding of the pyrimidine to the purineis referred to as “base pairing.” More specifically, A will hydrogenbond to T or U, and G will bond to C. “Complementary” refers to the basepairing that occurs between two distinct nucleic acid sequences or twodistinct regions of the same nucleic acid sequence.

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide and the DNA orRNA target. The oligonucleotide need not be 100% complementary to itstarget sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget DNA or RNA molecule interferes with the normal function of thetarget DNA or RNA, and there is sufficient degree of complementarity toavoid non-specific binding of the oligonucleotide to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ and/or Mg²⁺ concentration) of thehybridization buffer will contribute to the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed in Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chs. 9 and 11.

For purposes of the present disclosure, “stringent conditions” encompassconditions under which hybridization will occur if there is less than25% mismatch between the hybridization molecule and the target sequence.“Stringent conditions” can be further defined into particular levels ofstringency. Thus, as used herein, “moderate stringency” conditions arethose under which molecules with more than 25% mismatch will nothybridize; conditions of “medium stringency” are those under whichmolecules with more than 15% mismatch will not hybridize, and conditionsof “high stringency” are those under which sequence with more than 10%mismatch will not hybridize. Conditions of “very high stringency” arethose under which sequences with more than 6% mismatch will nothybridize.

In particular embodiments, stringent conditions are hybridization at 65°C., followed by sequential washes at 65° C. with 0.1×SSC/0.1% SDS for 40minutes.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein) has been substantially separated, produced apart from, orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, and proteins. Nucleicacid molecules and proteins that have been “isolated” include nucleicacid molecules and proteins purified by standard purification methods.The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell, as well as chemically synthesizednucleic acid molecules, proteins, and peptides.

Nucleic acid molecule: A polymeric form of nucleotides, which caninclude both sense and anti-sense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers of the above. A nucleotide refers toa ribonucleotide, deoxynucleotide, or a modified form of either type ofnucleotide. A “nucleic acid molecule” as used herein is synonymous with“nucleic acid” and “polynucleotide.” The term includes single- anddouble-stranded forms of DNA. A nucleic acid molecule can include eitheror both naturally occurring and modified nucleotides linked together bynaturally occurring and/or non-naturally occurring nucleotide linkages.

Nucleic acid molecules may be modified chemically or biochemically, ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications, such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., peptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). The term “nucleic acid molecule” also includes anytopological conformation, including single-stranded, double-stranded,partially duplexed, triplexed, hairpinned, circular, and padlockedconformations.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence is ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked with a coding sequence when thepromoter affects the transcription or expression of the coding sequence.When recombinantly produced, operably linked nucleic acid sequences aregenerally contiguous and, where necessary to join two protein-codingregions, in the same reading frame. However, elements need not becontiguous to be operably linked.

Promoter: A region of DNA that generally is located upstream (towardsthe 5′ region of a gene) that is needed for transcription. Promoterspermit the proper activation or repression of the gene which theycontrol. A promoter contains specific sequences that are recognized bytranscription factors. These factors bind to the promoter DNA sequencesand result in the recruitment of RNA polymerase, the enzyme thatsynthesizes the RNA from the coding region of the gene. In someembodiments, tissue-specific promoters are used in methods of theinvention, e.g., a pollen-specific promoter. A tissue-specific promoteris a DNA sequence that directs a higher level of transcription of anassociated gene in the tissue for which the promoter is specificrelative to the other tissues of the organism. Examples oftissue-specific promoters include tapetum-specific promoters;anther-specific promoters; pollen-specific promoters (see, e.g., U.S.Pat. No. 7,141,424, and International PCT Publication No. WO 99/042587);ovule-specific promoters; (see, e.g., U.S. Patent Application No.2001/047525 A1); fruit-specific promoters (See, e.g., U.S. Pat. Nos.4,943,674, and 5,753,475); and seed-specific promoters (see, e.g., U.S.Pat. Nos. 5,420,034, and 5,608,152). In some embodiments, developmentalstage-specific promoters are used in methods of the invention, e.g., apromoter active at a later stage in development.

Transformed: A virus or vector “transforms” or “transduces” a cell whenit transfers nucleic acid molecules into the cell. A cell is“transformed” by a nucleic acid molecule transduced into the cell whenthe nucleic acid molecule becomes stably replicated by the cell, eitherby incorporation of the nucleic acid molecule into the cellular genome,or by episomal replication. As used herein, the term “transformation”encompasses all techniques by which a nucleic acid molecule can beintroduced into such a cell. Examples include, but are not limited to,transfection with viral vectors, transformation with plasmid vectors,electroporation (Fromm et al. (1986) Nature 319:791-3), lipofection(Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7),microinjection (Mueller et al. (1978) Cell 15:579-85),Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad.Sci. USA 80:4803-7), direct DNA uptake, and microprojectile bombardment(Klein et al. (1987) Nature 327:70).

Transgene: An exogenous nucleic acid sequence. In one example, atransgene is a gene sequence (e.g., a herbicide-resistance gene), a geneencoding an industrially or pharmaceutically useful compound, or a geneencoding a desirable agricultural trait. In yet another example, thetransgene is an antisense nucleic acid sequence, wherein expression ofthe antisense nucleic acid sequence inhibits expression of a targetnucleic acid sequence. A transgene may contain regulatory sequencesoperably linked to the transgene (e.g., a promoter).

Vector: A nucleic acid molecule as introduced into a cell, therebyproducing a transformed cell. A vector can include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. Examples include, but are not limited to, aplasmid, cosmid, bacteriophage, or virus that carries exogenous DNA intoa cell. A vector can also include one or more genes, antisensemolecules, and/or selectable marker genes and other genetic elementsknown in the art. A vector can transduce, transform, or infect a cell,thereby causing the cell to express the nucleic acid molecules and/orproteins encoded by the vector. A vector optionally includes materialsto aid in achieving entry of the nucleic acid molecule into the cell(e.g., a liposome, protein coding, etc.).

Zn-Finger Nuclease-Mediated Excision of Transgenes from Plants

Disclosed herein are methods for producing a plant having decreasedtransgene escape, as well as plants produced by such methods, and plantmaterials derived therefrom, e.g., seeds. In one embodiment, the methodcomprises contacting a plant with a vector, wherein the vector includesone or more zinc finger nuclease(s) (ZFNs) operably linked to one ormore tissue-specific promoter(s) (e.g., a pollen-specific promoter).Expression of this vector results in the production of the ZFN(s) in thespecific tissue wherein its operably linked promoter is active. TheZFN(s) may be designed or engineered to recognize a cleavage sequencethat flanks a nucleic acid sequence, the excision of which is desired.Production of the ZFN(s), then, in the specific tissue wherein thepromoter is active, results in excision of the nucleic acid sequencebetween the cleavage sequences recognized by the ZFN(s), therebyproducing a nucleic acid sequence that contains a cleavage junction thatis free of a residual recognition sequence.

In another embodiment, the method comprises: contacting a plant with avector, wherein the vector includes one or more ZFN(s) operably linkedto a tissue-specific promoter; a gene of interest; optionally one ormore regulatory element(s) that may be operably linked to the gene ofinterest; and one or more cleavage sequences recognized by the ZFN(s)flanking the gene of interest and the one or more regulatory element(s).Expression of this vector results in the production of the ZFN(s) in thespecific tissue wherein its operably linked promoter is active.Production of the ZFN(s), then, in the specific tissue wherein thepromoter is active results in excision of the nucleic acid sequencebetween the cleavage sequences recognized by the ZFN(s), which includesthe gene of interest and, optionally, the one or more regulatoryelement(s).

In further embodiments, the method comprises contacting a plant with avector, wherein the vector includes one or more zinc finger nuclease(s)(ZFNs) operably linked to one or more promoter(s) active at a particularperiod of plant development (e.g., a promoter that drives expression ata relatively late stage of development). Expression of this vectorresults in the production of the ZFN(s) during the specific period ofdevelopment wherein its operably linked promoter is active. The ZFN(s)may be designed or engineered to recognize a cleavage sequence thatflanks a nucleic acid sequence, the excision of which is desired.Production of the ZFN(s) at the developmental stage wherein the promoteris active, results in excision of the nucleic acid sequence between thecleavage sequences recognized by the ZFN(s).

In still further embodiments, the method comprises: contacting a plantwith a vector, wherein the vector includes one or more ZFN(s) operablylinked to a promoter active at a particular period of plant development;a gene of interest; optionally one or more regulatory element(s) thatmay be operably linked to the gene of interest; and one or more cleavagesequences recognized by the ZFN(s) flanking the gene of interest and theone or more regulatory element(s). Expression of this vector results inthe production of the ZFN(s) during the specific period of developmentwherein its operably linked promoter is active. Production of the ZFN(s)during the specific period of development wherein its operably linkedpromoter is active, results in excision of the nucleic acid sequencebetween the cleavage sequences recognized by the ZFN(s), which includesthe gene of interest and the one or more regulatory element(s).

ZFN Nucleases

In particular embodiments, ZFNs are expressed from nucleic acidmolecules in transformed plants to direct the excision of nucleic acidsequences in the transformed plants. ZFNs may be used that target arecognition sequence engineered to flank a particular nucleic acidsequence (e.g., a transgene, gene of interest, or selectable markergene) or ZFNs may be designed to target a naturally occurring nucleicacid sequence flanking a particular nucleic acid sequence to be excised.The exquisite flexibility and specificity of the ZFN system provides alevel of control previously unachievable by known recombinase-mediatedgene excision strategies.

Recognition specificities of ZFNs can be easily manipulatedexperimentally. Wu et al. (2007) Cell. Mol. Life Sci. 64:2933-44.Randomization of the codons for zinc finger recognition residues allowsthe selection of new fingers that have high affinity for arbitrarilychosen DNA sequences. Furthermore, zinc fingers are natural DNA-bindingmolecules, and engineered zinc fingers have been shown to act on theirdesigned targets in living cells. Thus, nucleases based on zinc fingersare targetable to specific but arbitrary recognition sites.

The requirement for dimerization of cleavage domains of chimeric zincfinger nucleases imparts a high level of sequence specificity. Sinceeach set of three fingers binds nine consecutive base pairs, twochimeric nucleases effectively demand an 18 bp target if each zincfinger domain has perfect specificity. Any given sequence of this lengthis predicted to be unique within a single genome (assuming approximately10⁹ bp). Bibikova et al. (2001) Mol. Cell. Biol. 21(1):289-97; Wu et al.(2007), supra. Furthermore, additional fingers provide enhancedspecificity, Beerli et al. (1998) Proc. Natl. Acad. Sci. USA95:14628-33; Kim and Pabo (1998) Proc. Natl. Acad. Sci. USA 95:2812-7;Liu et al. (1997) Proc. Natl. Acad. Sci. USA 94:5525-30, so the numberof zinc fingers in each DNA-binding domain may be increased to provideeven further specificity. For example, specificity may be furtherincreased by using a pair of 4-finger ZFNs that recognize a 24 bpsequence. Urnov et al. (2005) Nature 435:646-51.

Key amino acids in ZFNs, at positions −1, 2, 3, and 6 relative to thestart of the α-helix, contribute most of the specific interactions bythe zinc finger motifs. Pavletich and Pabo (1991) Science 252:809-17;Shi and Berg (1995) Chem. Biol. 2:83-9. These amino acids can bechanged, while maintaining the remaining amino acids as a consensusbackbone, to generate ZFPs with different and/or novel sequencespecificities. See, e.g., Choo and Klug (1994) Proc. Natl. Acad. Sci.USA 91:11163-7; Desjarlais and Berg (1992) Proc. Natl. Acad. Sci. USA89:7345-9; Desjarlais and Berg (1993) Proc. Natl. Acad. Sci. USA90:2256-60; Greisman and Pabo (1997) Science 275:657-61; Isalan et al.(1998) Biochemistry 37:12026-33; Jamieson et al. (1994) Biochemistry33:5689-95; Rebar and Pabo (1994) Science 263:671-3; Segal et al. (1999)Proc. Natl. Acad. Sci. USA 96:2758-63; Wolfe et al. (1999) J. Mol. Biol.285:1917-34; Wu et al. (1995) Proc. Natl. Acad. Sci. USA 92:344-8.Moreover, at least two 3-finger ZFNs with different sequencespecificities can be designed, such that they collaborate to producecleavage. Smith et al. (2000), supra.

Design and selection approaches for constructing a ZFN of the inventionmay begin by determining one or more appropriate ZF motifs to recognizea specific nucleic acid sequence. Alternatively, a ZFN that recognizes aspecific nucleic acid sequence may be used to construct a nucleic acidmolecule comprising the specific nucleic acid sequence (e.g., whereinthe specific nucleic acid sequence flanks a gene of interest) and otherelements as needed. Design and various selection approaches for ZFPs,including the phage display method, have been reviewed. Mani et al.(2005), supra; Durai et al. (2005) Nucleic Acids Res. 33:5978-90; Isalanet al. (2001) Nat. Biotechnol. 19:656-60; Kandavelou et al. (2005) Nat.Biotechnol. 23:686-87; Pabo et al. (2001) Annu. Rev. Biochem. 70:313-40;Segal et al. (2003) Biochemistry 42:2137-48. Any design and/or selectionapproach known in the art may be used to arrive at a ZFN for use inembodiments of the present invention. For example, cell-based selectionstrategies using bacterial one-hybrid and two-hybrid systems may be usedto produce highly specific ZFPs. Durai et al. (2006) Comb. Chem. HighThroughput Screen. 9:301-11; Hurt et al. (2003) Proc. Natl. Acad. Sci.USA 100:12271-6; Joung et al. (2000) Proc. Natl. Acad. Sci. USA97:7382-7. Highly specific ZFPs can also be obtained by directed domainshuffling and cell-based selection, which offers a general approach foroptimizing multi-finger ZFPs. Hurt et al. (2003), supra.

A wealth of data based on design and phage display methodologies isavailable for ZF modules that specifically recognize 5′ GNN 3′ and 5′ANN 3′ triplets, and to a lesser extent, the ZF motif preferences for 5′CNN 3′ and 5′ TNN 3′ triplets are known. See, e.g., Durai et al. (2005),supra; Dreier et al. (2001) J. Biol. Chem. 276:29466-78; Dreier et al.(2005) J. Biol. Chem. 280:35588-97; Dreier et al. (2000) J. Mol. Biol.303:489-502; Liu et al. (2002) J. Biol. Chem. 277:3850-6. Currently, twoWeb-based ZF design software packages are available (e.g., atzincfingertools.org). The foregoing renders nearly all genes encoded ina genome amenable to ZFN-mediated gene targeting. Katada and Komiyama(2009) Chembiochem. 10(8):1279-88.

In particular embodiments, a ZFN is used that binds the HIV co-receptorCCR5. Perez et al. (2008) Nat. Biotechnol. 26:808-16. This ZFN is termedthe “CCR5 ZFN.” In particular embodiments, the CCR5 ZFN coding regioncomprises: the opaque-2 nuclear localization sequence (Maddaloni et al.(1989) Nucleic Acids Res. 17(18):7532); the r162y11 zinc finger bindingdomain, the Fold nuclease domain (Looney et al. (1989) Gene 80:193-208);a T2A stutter sequence (Mattion et al. (1996) J. Virol. 70:8124-7)derived from the Thesoa assigna virus; a second opaque-2 nuclearlocalization sequence, the 168FA vE zinc finger binding domain; and asecond FokI nuclease domain.

Nucleic Acid Molecules

In some embodiments, the method includes crossing a first plant havingone or more genes of interest (which may confer a desirable trait orphenotype), such as two or more genes of interest, with a second plant.The second plant may also have one or more genes of interest. The firstplant may include a vector, wherein the vector includes a promoteroperably linked to one or more gene(s) of interest. The promoter may bea constitutive or inducible promoter. The nucleic acid sequence encodinga gene(s) of interest may be flanked by ZFN recognition sites.Optionally, the promoter operably linked to the gene(s) of interest, andany additional nucleic acid sequences (e.g., regulatory sequences), mayalso be flanked by ZFN recognition sites. The second plant may includeanother vector, which may include a tissue-specific ordevelopment-specific promoter operably linked to a nucleic acid sequenceencoding a ZFN. The vectors may be stably integrated into the genomes ofboth plants. After crossing the first and second plants, thetissue-specific or development-specific promoter specifically drives theexpression of the ZFN in the resulting progeny of such a cross.Expression of the ZFN in these progeny leads to excision of nucleic acidsequences flanked by the ZFN recognition sites, thereby reducing oreliminating the gene of interest, and optionally additional sequences(such as selectable marker genes) in specific tissues and/or stages ofdevelopment of the progeny. In some embodiments, the ZFN recognitionsites may be further flanked by homologous nucleic acid sequences tofurther promote homologous DNA recombination.

A gene of interest will typically be operably linked to one or moreplant promoter(s) driving expression of the gene in an amount sufficientto confer a desired trait or phenotype. Promoters suitable for this andother uses are well known in the art. Non-limiting examples describingsuch promoters include U.S. Pat. No. 6,437,217 (maize RS81 promoter);U.S. Pat. No. 5,641,876 (rice actin promoter); U.S. Pat. No. 6,426,446(maize RS324 promoter); U.S. Pat. No. 6,429,362 (maize PR-1 promoter);U.S. Pat. No. 6,232,526 (maize A3 promoter); U.S. Pat. No. 6,177,611(constitutive maize promoters); U.S. Pat. Nos. 5,322,938, 5,352,605,5,359,142, and 5,530,196 (35S promoter); U.S. Pat. No. 6,433,252 (maizeL3 oleosin promoter); U.S. Pat. No. 6,429,357 (rice actin 2 promoter,and rice actin 2 intron); U.S. Pat. No. 5,837,848 (root-specificpromoter); U.S. Pat. No. 6,294,714 (light-inducible promoters); U.S.Pat. No. 6,140,078 (salt-inducible promoters); U.S. Pat. No. 6,252,138(pathogen-inducible promoters); U.S. Pat. No. 6,175,060 (phosphorousdeficiency-inducible promoters); U.S. Pat. No. 6,388,170 (bidirectionalpromoters); U.S. Pat. No. 6,635,806 (gamma-coixin promoter); and U.S.patent application Ser. No. 09/757,089 (maize chloroplast aldolasepromoter). Additional promoters include the nopaline synthase (NOS)promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. USA 84(16):5745-9);the octopine synthase (OCS) promoter (which is carried on tumor-inducingplasmids of Agrobacterium tumefaciens); the caulimovirus promoters suchas the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al.(1987) Plant Mol. Biol. 9:315-24); the CaMV 35S promoter (Odell et al.(1985) Nature 313:810-2; the figwort mosaic virus 35S-promoter (Walkeret al. (1987) Proc. Natl. Acad. Sci. USA 84(19):6624-8); the sucrosesynthase promoter (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA87:4144-8); the R gene complex promoter (Chandler et al. (1989) PlantCell 1:1175-83); the chlorophyll alb binding protein gene promoter;CaMV35S (U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and 5,530,196);FMV35S (U.S. Pat. Nos. 6,051,753, and 5,378,619); a PC1SV promoter (U.S.Pat. No. 5,850,019); the SCP1 promoter (U.S. Pat. No. 6,677,503); andAGRtu.nos promoters (GenBank Accession No. V00087; Depicker et al.(1982) J. Mol. Appl. Genet. 1:561-73; Bevan et al. (1983) Nature304:184-7), and the like.

Additional genetic elements that may optionally be operably linked to agene of interest include sequences coding for transit peptides. Forexample, incorporation of a suitable chloroplast transit peptide, suchas the A. thaliana EPSPS CTP (Klee et al. (1987) Mol. Gen. Genet.210:437-42), and the Petunia hybrida EPSPS CTP (della-Cioppa et al.(1986) Proc. Natl. Acad. Sci. USA 83:6873-7) has been shown to targetheterologous EPSPS protein sequences to chloroplasts in transgenicplants. Dicamba monooxygenase (DMO) may also be targeted tochloroplasts, as described in International PCT Publication No. WO2008/105890.

Additional genetic elements that may optionally be operably linked to agene of interest also include 5′ UTRs located between a promotersequence and a coding sequence that function as a translation leadersequence. The translation leader sequence is present in the fullyprocessed mRNA upstream of the translation start sequence. Thetranslation leader sequence may affect processing of the primarytranscript to mRNA, mRNA stability, and/or translation efficiency.Examples of translation leader sequences include maize and petunia heatshock protein leaders (U.S. Pat. No. 5,362,865), plant virus coatprotein leaders, plant rubisco leaders, and others. See, e.g., Turnerand Foster (1995) Molecular Biotech. 3(3):225-36. Non-limiting examplesof 5′ UTRs include GmHsp (U.S. Pat. No. 5,659,122); PhDnaK (U.S. Pat.No. 5,362,865); AtAnt1; TEV (Carrington and Freed (1990) J. Virol.64:1590-7); and AGRtunos (GenBank Accession No. V00087; and Bevan et al.(1983) Nature 304:184-7).

Additional genetic elements that may optionally be operably linked to agene of interest also include 3′ non-translated sequences, 3′transcription termination regions, or poly-adenylation regions. Theseare genetic elements located downstream of a polynucleotide molecule,and include polynucleotides that provide polyadenylation signal, and/orother regulatory signals capable of affecting transcription, mRNAprocessing, or gene expression. The polyadenylation signal functions inplants to cause the addition of polyadenylate nucleotides to the 3′ endof the mRNA precursor. The polyadenylation sequence can be derived fromthe natural gene, from a variety of plant genes, or from T-DNA genes. Anon-limiting example of a 3′ transcription termination region is thenopaline synthase 3′ region (nos 3; Fraley et al. (1983) Proc. Natl.Acad. Sci. USA 80:4803-7). An example of the use of different 3′nontranslated regions is provided in Ingelbrecht et al., (1989) PlantCell 1:671-80. Non-limiting examples of polyadenylation signals includeone from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al. (1984)EMBO J. 3:1671-9) and AGRtu.nos (GenBank Accession No. E01312).

Plant Transformation

Any of the techniques known in the art for introduction of transgenesinto plants may be used to produce a transformed plant according to theinvention. Suitable methods for transformation of plants are believed toinclude virtually any method by which DNA can be introduced into a cell,such as: by electroporation as illustrated in U.S. Pat. No. 5,384,253;by microprojectile bombardment, as illustrated in U.S. Pat. Nos.5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865; byAgrobacterium-mediated transformation as illustrated in U.S. Pat. Nos.5,635,055, 5,824,877, 5,591,616; 5,981,840, and 6,384,301; and byprotoplast transformation, as set forth in U.S. Pat. No. 5,508,184, etc.Through the application of techniques such as these, the cells ofvirtually any plant species may be stably transformed, and these cellsmay be developed into transgenic plants by techniques known to those ofskill in the art. Techniques that may be particularly useful in thecontext of cotton transformation are disclosed in U.S. Pat. Nos.5,846,797, 5,159,135, 5,004,863, and 6,624,344; techniques fortransforming Brassica plants in particular are disclosed, for example,in U.S. Pat. No. 5,750,871; techniques for transforming soybean aredisclosed, for example, in U.S. Pat. No. 6,384,301; and techniques fortransforming corn are disclosed, for example, in U.S. Pat. Nos.7,060,876, 5,591,616, and International PCT Publication WO 95/06722.

After effecting delivery of exogenous DNA to recipient cells, the nextsteps generally concern identifying the transformed cells for furtherculturing and plant regeneration. In order to improve the ability toidentify transformants, one may desire to employ a selectable orscreenable marker gene with the transformation vector used to generatethe transformant. In this case, the potentially transformed cellpopulation can be assayed by exposing the cells to a selective agent oragents, or the cells can be screened for the desired marker gene trait.

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In some embodiments, any suitableplant tissue culture media (e.g., MS and N6 media) may be modified byincluding further substances, such as growth regulators. Tissue may bemaintained on a basic media with growth regulators until sufficienttissue is available to begin plant regeneration efforts, or followingrepeated rounds of manual selection, until the morphology of the tissueis suitable for regeneration (e.g., at least 2 weeks), then transferredto media conducive to shoot formation. Cultures are transferredperiodically until sufficient shoot formation has occurred. Once shootsare formed, they are transferred to media conducive to root formation.Once sufficient roots are formed, plants can be transferred to soil forfurther growth and maturity.

To confirm the presence of a gene of interest (e.g., a transgene) in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example: molecular biological assays, such as Southern andNorthern blotting and PCR; biochemical assays, such as detecting thepresence of a protein product, e.g., by immunological means (ELISAand/or Western blots) or by enzymatic function; plant part assays, suchas leaf or root assays; and analysis of the phenotype of the wholeregenerated plant.

Cultivation and Use of Transgenic Plants

A plant exhibiting nucleic acid excision according to the presentinvention may have one or more desirable traits, such as two or moredesirable traits. Such traits can include, for example: resistance toinsects and other pests and disease-causing agents; tolerances toherbicides; enhanced stability, yield, or shelf-life; environmentaltolerances; pharmaceutical production; industrial product production;and nutritional enhancements. The desirable traits may be conferred bygenes flanked by nucleic acid sequence recognized by ZFN(s) expressed inthe plant exhibiting the desirable traits, such that expression of theZFN(s) in the plant decreases or eliminates transmission of the trait,through containment of its underlying gene, to other plants orsubsequent generations of the plant. Thus, in one embodiment, thedesired trait can be due to the presence of a transgene(s) in the plant,which may be flanked by ZFN recognition sequences. In an additionalembodiment, the desirable trait can be obtained through conventionalbreeding, which trait may be conferred by one or more genes flanked byZFN recognition sequences.

A plant exhibiting nucleic acid excision according to the invention maybe any plant capable of being transformed with a nucleic acid moleculeof the invention. Accordingly, the plant may be a dicot or monocot.Non-limiting examples of dicotyledonous plants usable in the presentmethods include alfalfa, beans, broccoli, cabbage, carrot, cauliflower,celery, Chinese cabbage, cotton, cucumber, eggplant, lettuce, melon,pea, pepper, peanut, potato, pumpkin, radish, rapeseed, spinach,soybean, squash, sugarbeet, sunflower, tobacco, tomato, and watermelon.Non-limiting examples of monocotyledonous plants usable in the presentmethods include corn, onion, rice, sorghum, wheat, rye, millet,sugarcane, oat, triticale, switchgrass, and turfgrass.

Plants exhibiting nucleic acid excision according to the invention maybe used or cultivated in any manner, wherein transmission of the excisednucleic acid sequence to other plants is undesirable. Accordingly, GMplants that have been engineered to, inter alia, have one or moredesired traits, may be transformed with nucleic acid molecules accordingto the invention, and cropped and cultivated by any method known tothose of skill in the art.

EXAMPLES

The following examples are included to illustrate embodiments of theinvention. It will be appreciated by those of skill in the art that thetechniques disclosed in the Examples represent techniques discovered bythe inventors to function well in the practice of the invention.However, those of skill in the art will, in light of the presentdisclosure, can appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the scope of the invention. Morespecifically, it will be apparent that certain agents that are bothchemically and physiologically related may be substituted for the agentsdescribed herein, while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the scope of the invention as definedby the appended Claims.

Example I: Plasmid Design and Construction

A target construct containing a target reporter gene expression cassetteflanked by zinc finger binding sites (pDAS5380) and an excisionconstruct containing a zinc finger nuclease gene expression cassette(pDAS5381) were designed and constructed. The constructs were designedto be transformed separately into tobacco. Target reporter gene excisionwas carried out by crossing the two tobacco lines, wherein a functionalzinc finger nuclease recognized the zinc finger binding sites flankingthe target reporter gene cassette and cleaved the genomic DNA. Crossingthe plant lines containing the target reporter gene construct with theplant line containing the excision construct resulted in theremoval/deletion of the reporter gene from the plant genome.

Construction and Design of Target Construct pDAS5380.

pDAS5380 (FIG. 1) was constructed as a binary plasmid vector. Thisconstruct contains the following plant transcription unit (PTU)expression cassettes and genetic elements: RB7 MAR ((Matrix AttachmentRegion (Thompson et al. (1997) WO9727207)):: CCR5 binding site repeated4× (Perez et al. (2008) Nat. Biotechnol. 26:808-16):: AtuORF1 3′ UTR(Agrobacterium tumefaciens open reading frame-1, 3′ untranslated region(Huang et al. (1990) J. Bacteriol. 172:1814-22))/GUS (β-D-glucuronidase(Jefferson (1989) Nature 342:837-8))/AtUbi10 (Arabidopsis thalianaubiquitin-10 promoter (Callis et al. (1990) J. Biol. Chem.265:12486-93)):: CCR5 Binding Site repeated 4×:: AtAct2 (A. thalianaactin-2 promoter (An et al. (1996) Plant J. 10:107-21))/Turbo GFP(turbo-green fluorescence protein (Evdokimov et al. (2006) EMBO Rep.7(10):1006-12))/Atu ORF23 3′ UTR (A. tumefaciens open reading frame-23,3′ untranslated region (Gelvin et al. (1987) EP222493)):: AtUbi10/PAT(phosphinothricin acetyl transferase (Wohlleben et al. (1988) Gene70:25-37))/Atu ORF1 3′ UTR. The GUS PTU expression cassette was placedin trans to the GFP and PAT PTU expression cassettes. In addition, theGUS PTU expression cassette was flanked by CCR5 zinc finger nucleasebinding sites. This sequence (SEQ ID NO:1) was repeated 4× directlyupstream and downstream of the GUS PTU expression cassette. Thelocations of the zinc finger binding sites are identified in FIG. 1 as“CCR5 BINDING SITE.” These sites are recognized and bound by the zincfinger nuclease protein encoded by excisor construct, pDAS5381. Theassembly of this binary vector was completed using standard molecularbiology techniques. The final plasmid was confirmed via restrictionenzyme digestion and DNA sequencing.

Construction and Design of Excisor Construct, pDAS5381.

A binary plasmid containing a zinc finger nuclease gene that wasspecifically designed to bind the CCR5 binding site (SEQ ID NO:2) wasdesigned and constructed as described in Perez et al., (2008) NatureBiotechnol. 26:808-16. pDAS5381 (FIG. 2) contains the following PTUexpression cassettes: CsVMV (Cassava Vein Mosaic Virus promoter(Verdaguer et al. (1996) Plant Mol. Biol. 31:1129-39))/CCR5 zinc fingernuclease coding region (containing: the opaque-2 nuclear localizationsequence (Maddaloni et al. (1989) Nucleic Acids Res. 17(18):7532); ther162y11 zinc finger binding domain; the Fold nuclease domain (Looney etal. (1989) Gene 80:193-208); a T2A stutter sequence (Mattion et al.(1996) J. Virol. 70:8124-7) derived from the Thesoa assigna virus; asecond opaque-2 nuclear localization sequence; the 168GA vE zinc fingerbinding domain; and a second FokI nuclease domain)/Atu ORF23 3′ UTR::AtUbi3 promoter (A. thaliana ubiquitin-3 promoter (Callis et al. (1995)Genetics 139(2):921-39))/HPTII (hygromycin phosphotransferase II (Gritzet al. (1983) Gene 25(2-3):179-88))/Atu ORF24 3′ UTR (A. tumefaciensopen reading frame-24, 3′ untranslated region (Gelvin et al. (1987)EP222493)). The assembly of this binary vector was completed usingstandard molecular biology techniques. The final plasmid was confirmedvia restriction enzyme digestion and DNA sequencing.

Example II: Agrobacterium-Mediated Plant Transformation

Transformation of Agrobacterium with pDAS5380 and pDAS5381.

Electrocompetent A. tumefaciens (strain LBA4404) cells were obtainedfrom Invitrogen (Carlsbad, Calif.) and transformed using anelectroporation method adapted from Weigel and Glazebrook (2002) “How toTransform Arabidopsis,” in Arabidopsis: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., U.S.A. Transformedcolonies were obtained on yeast extract peptone media (YEP) containingspectinomycin (50 μg/mL) and streptomycin (125 μg/mL) and confirmed viarestriction enzyme digestion. Clones which exhibited the correctrestriction enzyme banding patterns were stored as glycerol stocks at−80° C.

Agrobacterium—Mediated Transformation of Nicotiana tabacum.

Tobacco (cv. Petit Havana) leaf discs were transformed using A.tumefaciens (strain LBA4404) containing pDAS5381 and pDAS5380. Singlecolonies of Agrobacterium containing these plasmids were inoculated into4 mL of YEP containing spectinomycin (50 μg/mL) and streptomycin (125μg/mL) and incubated overnight at 28° C. on a shaker at 190 rpm. The 4mL seed culture was subsequently used to inoculate a 25 mL culture ofYEP media containing spectinomycin (50 μg/mL) and streptomycin (125μg/mL) grown in a 125 mL baffled Erlenmeyer flask. This culture wasincubated at 28° C. shaking at 190 rpm until it reached an OD₆₀₀ of˜1.2. Ten mL of Agrobacterium suspension was placed into sterile 60×20mm Petri dishes.

Twenty-five freshly cut leaf discs (0.5 cm²) cut from plants asepticallygrown on MS medium (Phytotechnology Labs, Shawnee Mission, Kans., #M524)with 30 g/L sucrose in PhytaTrays™ (Sigma, St. Louis, Mo.) were soakedin 10 mL of overnight culture of Agrobacterium for a few minutes,blotted dry on sterile filter paper, and then placed onto the samemedium with the addition of 1 mg/L indoleacetic acid and 1 mg/Lbenzyamino purine. Following 48 hours of co-cultivation, leaf discsco-cultivated with Agrobacterium harboring pDAS5380 were transferred tothe same medium with 5 mg/L Basta® and 250 mg/L cephotaxime. Leaf discsco-cultivated with Agrobacterium harboring pDAS5381 were transferred tothe same medium with 10 mg/L hygromycin and 250 mg/L cephotaxime. After3 weeks, individual T₀ plantlets were transferred to either MS mediumwith 10 mg/L Basta® and 250 mg/L cephotaxime for pDAS5380, or with 10mg/L hygromycin and 250 mg/L cephotaxime for pDAS5381, an additional 3weeks prior to transplanting to soil and transfer to the greenhouse.

Copy Number, Full Length PTU and Expression Analysis of T₀ Plants.

Copy Number Assay.

Invader® and hydrolysis probe assays were performed to screen samples ofBasta®-resistant plants to identify those that contained single copyintegration of the T-DNA in pDAS5380 and pDAS5381. Detailed analysis wasconducted using primers and probes specific to gene expressioncassettes. Single copy events were identified for additional analysis.

Tissue samples were collected in 96-well plates and lyophilized for 2days. Tissue maceration was performed with a Kleco™ tissue pulverizerand tungsten beads (Visalia, Calif.). Following tissue maceration, thegenomic DNA was isolated in high-throughput format using the DNeasy 96Plant Kit™ (Qiagen, Germantown, Md.) according to the manufacturer'ssuggested protocol. Genomic DNA was quantified by Quant-IT Pico GreenDNA assay Kit™ (Molecular Probes, Invitrogen, Carlsbad, Calif.).Quantified genomic DNA was adjusted to 9 ng/μL for the Invader® assay orto 5 ng/μL for the hydrolysis probe assay using a Biorobot3000™automated liquid handler (Qiagen, Germantown, Md.).

Custom Invader® assays were developed for PAT gene analysis in tobaccoby Hologic (Madison, Wis.). The genomic DNA samples (7.5 μL at 9 ng/μL)were first denatured in 96-well plate format by incubation at 95° C. for10 minutes and then cooled on ice. Next, 7.5 μL of master mix (3 μL ofprobe mix for pat and an internal reference gene (phenylalanine ammoniumlyase (palA); GenBank ID: AB008199), 3.5 μL Cleavase® XI FRET mix, and 1μL of Cleavase® XI Enzyme/MgCl₂ solution) were added to each well andthe samples were overlaid with mineral oil. Plates were sealed andincubated at 63° C. for 1 hour in a BioRad Tetrad® thermocycler. Plateswere cooled to ambient temperature before being read on a fluorescenceplate reader. All plates contained 1 copy, 2 copy and 4 copy standardsas well as wild-type control samples and blank wells containing nosample. Readings were collected for both FAM (λ, 485-528 nm) and RED (λ,560-620 nm) channels, and from these the fold over zero (i.e.,background) for each channel was determined for each sample by thesample raw signal divided by no template raw signal. From this data, astandard curve was constructed and the best fit determined by linearregression analysis. Using the parameters identified from this fit, theapparent pat copy number was then estimated for each sample.

Transgene copy number determination by hydrolysis probe assay, analogousto TaqMan® assay, was performed by real-time PCR using the LightCycler®480 system (Roche Applied Science, Indianapolis, Ind.). Assays weredesigned for HPTII, PAT and the internal reference gene phenylalanineammonium lyase (palA) using LightCycler® Probe Design Software 2.0. Foramplification, LightCycler® 480 Probes Master mix (Roche AppliedScience, Indianapolis, Ind.) was prepared at 1× final concentration in a10 μL volume multiplex reaction containing 0.4μM of each primer and 0.2μM of each probe (Table 1). A two-step amplification reaction wasperformed with an extension at 58° C. for 38 seconds with fluorescenceacquisition. All samples were run in triplicate and the averaged Cyclethreshold (Ct) values were used for analysis of each sample. Analysis ofreal time PCR data was performed using LightCycler® software release 1.5using the relative quant module and is based on the AACt method. Forthis, a sample of genomic DNA from a single copy calibrator and known 2copy check were included in each run (identical to those used forInvader® assays above).

TABLE 1Primer and probe Information for hydrolysis probe assay of PAT, HPTII,and internal reference (palA). Primer Name Sequence Detection TQPATSSEQ ID NO: 3; 5′ ACAAGAGTGGATTGATGATCTAGAGAGGT 3′ TQPATASEQ ID NO: 4; 5′ CTTTGATGCCTATGTGACACGTAAACAGT 3′ TQPATFQSEQ ID NO: 5; 5′ Cy5 CY5-GGTGTTGTGGCTGGTATTGCTTACGCTGG-BHQ2 3′ TQPALSSEQ ID NO: 6; 5′ TACTATGACTTGATGTTGTGTGGTGACTGA 3′ TQPALASEQ ID NO: 7; 5′ GAGCGGTCTAAATTCCGACCCTTATTTC 3′ TQPALFQSEQ ID NO: 8; 5′ 6FAM 6FAM-AAACGATGGCAGGAGTGCCCTTTTTCTATCAAT-BHQ1 3′HPT2S SEQ ID NO: 9; 5′ ACACTACATGGCGTGATTT 3′ HPT2A SEQ ID NO: 10; 5′AGCATCAGCTCATCGAGA 3′ HPTFQ SEQ ID NO: 11; 5′Cy5/ACTGTGATGGACGACACCG/3BHQ2/3′ Cy5

Full Length PTU Assay Via Southern Blot Analysis.

Southern blot analysis was used to establish the integration pattern ofthe inserted DNA fragment and identify pDAS5380 and pDAS5381 eventswhich contained a full length PTU. Data were generated to demonstratethe integration and integrity of the transgenes inserted into thetobacco genome. Southern blot data was used to identify simpleintegration of an intact copy of the T-DNA from pDAS5380 and pDAS5381.Detailed Southern blot analysis was conducted using probes specific togene expression cassettes. The hybridization of these probes withgenomic DNA that had been digested with specific restriction enzymesidentified genomic DNA fragments of molecular weights, the patterns ofwhich could be analyzed to identify events for advancement to T₁. Theseanalyses also showed that the plasmid fragment had been inserted intotobacco genomic DNA without rearrangements of the PTU.

Tissue samples were collected in 50 mL conical tubes (Fisher Scientific,Pittsburgh, Pa.) and lyophilized for 2 days. Tissue maceration wasperformed with a paint mixer tissue pulverizer and tungsten beads.Following tissue maceration, the genomic DNA was isolated using theDNeasy™ Plant Maxi Kit (Qiagen, Germantown, Md.) according tomanufacturer suggested protocol. Purified genomic DNA was precipitatedand resuspended in 500 μL TE buffer. The genomic DNA was furtherpurified using the Qiagen Genomic Tips™ kit. Genomic DNA was quantifiedby Quant-IT Pico Green™ DNA assay kit (Molecular Probes, Invitrogen,Carlsbad, Calif.). Quantified genomic DNA was adjusted to 8 μg in aconsistent volume.

For each sample, 8 μg of genomic DNA was thoroughly digested with therestriction enzymes MfeI and NsiI (New England Biolabs, Beverley,Mass.). Samples were incubated at 37° C. overnight. The digested DNA wasconcentrated by precipitation with Quick Precipitation Solution™ (EdgeBiosystems, Gaithersburg, Md.) according to the manufacturer's suggestedprotocol. The genomic DNA was then resuspended in 25 μL of water at 65°C. for 1 hour. Resuspended samples were loaded onto a 0.8% agarose gelprepared in 1×TAE and electrophoresed overnight at 1.1 V/cm in 1×TAEbuffer. The gel was sequentially subjected to denaturation (0.2 MNaOH/0.6 M NaCl) for 30 minutes, and neutralization (0.5 M Tris-HCl (pH7.5)/1.5 M NaCl) for 30 minutes.

Transfer of DNA fragments was performed by passively wicking 20×SSCsolutions overnight through the gel onto treated Immobilon™ NY+ transfermembrane (Millipore, Billerica, Mass.) by using a chromatography paperwick and paper towels. Following transfer, the membrane was brieflywashed with 2×SSC, cross-linked with the Stratalinker™ 1800 (Stratagene,LaJolla, Calif.), and vacuum baked at 80° C. for 3 hours.

Blots were incubated with pre-hybridization solution (Perfect Hyb Plus™,Sigma, St. Louis, Mo.) for 1 hour at 65° C. in glass roller bottlesusing a model 400 hybridization incubator (Robbins Scientific,Sunnyvale, Calif.). Probes were prepared from a PCR fragment containingthe entire coding sequence. The PCR amplicon was purified using QIAEXII™ gel extraction kit and labeled with α³²P-dCTP via the Random RTPrime IT™ labeling kit (Stratagene, La Jolla, Calif.). Blots werehybridized overnight at 65° C. with denatured probe added directly tohybridization buffer to approximately 2 million counts per blot per mL.Following hybridization, blots were sequentially washed at 65° C. with0.1×SSC/0.1% SDS for 40 minutes. Finally, the blots were exposed tochemiluminescent film (Roche Diagnostics, Indianapolis, Ind.) and imagedusing a Molecular Dynamics Storm860™ imaging system.

Expected and observed fragment sizes with a particular digest and probe,based on the known restriction enzyme sites of the pDAS5380 or pDAS5381fragment, are indicated in FIGS. 3 and 4. The Southern blot analysescompleted in this study were used to identify events that containedfull-length intact PTUs from plasmids pDAS5380 or pDAS5381 that wereinserted into the tobacco genome (FIGS. 3 and 4, respectively).

GUS Expression Assay.

To test whether the pDAS5380 transgenic plants contained a functionalGUS PTU expression cassette, leaf samples were harvested and stainedhistochemically for GUS expression. Leaf discs (˜0.25 cm²) were cut andplaced in a 24-well tray (1 leaf disc per well) containing 250 μL of GUSassay solution (Jefferson (1989) Nature 342:837-8). The 24-well dish waswrapped with Nescofilm® (Fisher Scientific, Pittsburgh, Pa.) andincubated at 37° C. for 24 hours. After 24 hours, the GUS assay solutionwas removed from each well and replaced with 250 μL of 100% ethanol. Thedish was wrapped with Nescofilm® and incubated at room temperature for2-3 hours. The ethanol was removed and replaced with fresh ethanol. Theleaf discs were then viewed under a dissecting microscope. Leaf discswhich were stained blue were scored as containing a functional GUS PTUexpression cassette.

GFP Expression Assay.

Tobacco leaf samples were analyzed for GFP expression using ELISA.Plates were coated with a purified rabbit anti-GFP antibody overnight at4° C. The day of analysis, plates were blocked with 0.5% BSA in PBST.Duplicated leaf samples were extracted by bead beating frozen leafpieces with 2 stainless steel beads in a Kleco™ tissue grinder for 3minutes at maximum speed. The samples were centrifuged at 3000 rcf for10 minutes and the supernatants collected. Extract samples were loadedonto ELISA plates at 1:5 and 1:50 dilutions. An E. coli recombinant GFPstandard curve was run on each plate with concentrations from 12.5 ng/mLto 0.195 ng/mL. The standards and samples were incubated on the ELISAplates for 1 hour. Plates were washed and a horseradish peroxidaseconjugated rabbit anti-GFP antibody was added. Following 1 hourincubation, the plates were washed and substrate was added. Color wasallowed to develop before stopping the reaction with H₂SO₄. Absorbancewas read on a plate reader at 450 nm with a 650 nm reference filter. Aquadratic standard curve was generated by fitting concentration of theE. coli standard against OD. Concentrations of unknown samples weredetermined by linear regression.

Selection of T₀ Plants for Target T₁ Production.

A total of 68 Baste-resistant, GUS+/GFP+ plants were regenerated and 38plants were found to have 1-2 transgene copies based on PAT Invader®assay. Southern analysis identified 14 single-copy events, of which 8displayed bands consistent with intact PAT, GUS and GFP PTUs. ThreepDAS5380 events displaying single copy, full length PTU, and expressingGUS and GFP, pDAS5380-3, pDAS5380-18 and pDAS5380-46, wereself-pollination to produce T₁ seed.

FokI Expression Assay.

Quantitative Real-Time PCR (qRT-PCR) was used to quantify the mRNAexpression of the zinc finger nuclease in T₀ tobacco plants transformedwith pDAS5381. The assay was developed to quantify the relative FoldmRNA expression from tobacco leaf samples by normalizing these levelsagainst mRNA expression from input mRNA. The normalization of the FokImRNA against total mRNA permits comparison of Fold expression betweendifferent samples, and can be used to identify events that appear to behighly expressing. The relative ZFN expression is listed in Table 1.1.

TABLE 1.1 Quantification of mRNA expression of the zinc finger nucleasein T₀ tobacco plants transformed with pDAS5381. Relative ZFN Standard T0Event Expression* Deviation % CV pDAS5381-14 3.21 1.56 36.0 pDAS5381-1841.30 1.56 3.8 pDAS5381-30 8.39 0.86 10.3 pDAS5381-39 17.70 1.92 10.8pDAS5381-49 47.55 1.79 3.8 pDAS5381-54 4.45 0.57 12.8 pDAS5381-56 11.732.5 21.3 *qRT-PCR for Fok1 mRNA normalized to total RNA. Mean of 4replicate samples.

Leaf material from T₀ tobacco plants that had been transformed withpDAS5381 was collected and placed on ice. Total RNA was isolated usingQiagen's RNeasy® Plant Mini Kit (Qiagen, Germantown, Md.). Total mRNAwas treated with RNase-free DNase per the manufacturer's recommendationto remove any contaminating DNA that might amplify during quantitativeRT-PCR. First strand synthesis was set up according to the SuperscriptIII™ Reverse Transcriptase Enzyme (Invitrogen, Carlsbad, Calif.)manufacturer's instructions and primed using random hexamers. Thesynthesized cDNA strands were diluted in water at ratios of 1:10 and1:50. Each aliquot was stored at −20° C.

The qRT-PCR reaction was completed as follows: forward primer FokI_UPL_F(SEQ ID NO:12), reverse primer FokI_UPL_R (SEQ ID NO:13), probe UPL#130(cat #04693663001, Roche, Indianapolis, Ind.), 1× LC480 Probes MasterBuffer (Roche Diagnostic, Indianapolis, Ind.), and 1.5 μL of synthesizedcDNA in a 15 μL reaction. Serial dilutions of the synthesized cDNA weremade and assayed in repetition. The cocktail was amplified usingLightCycler® 480 Probes Master kit #04707494001 (Roche Diagnostics,Indianapolis, Ind.). A 96-well microplate was demarcated and labeled,13.5 μL of master mix was added per well. A sealing foil was gentlyattached to the microplate. The plate was centrifuged for 1 minute at3,000 rpm in a Qiagen microplate centrifuge. The sealing foil wasremoved and 1.5 μL of thawed, diluted synthesized cDNA strands wereadded. A foil seal was firmly affixed to the plate and centrifuged aspreviously described. A PCR program was run as follows: i) activate 95°C. for 5 minutes; ii) denature 95° C. for 10 sec (@ 4.8° C./sec); iii)anneal/extend 60° C. for 25 sec (@ 2.5° C./sec); iv) acquire 72° C. for1 sec (@ 4.8° C./sec); steps ii-iv were repeated 45 more times; vi) coolto 38° C. for 5 sec.

A qRT-PCR assay for quantifying the mRNA expression of the internalreference gene was completed as another method to normalize the zincfinger nuclease mRNA expression. The actin qRT-PCR reaction wascompleted as follows: forward primer BY2ACT89S (SEQ ID NO:14), reverseprimer BY2ACT89A (SEQ ID NO:15), probe BYACTFQ (SEQ ID NO:18), 1× LC480Probes Master Buffer, and 2.0 μL of synthesized cDNA, in a 10 μLreaction. Serial dilutions of the synthesized cDNA were made and assayedin repetition. In addition, 2 μL of plasmid DNA copy number standardswere added to separate wells in a dilution series from lowest to highestconcentrations, and these standards were compared to the actin cDNA(synthesized from total mRNA) to quantify the copy number. Actin DNAcopy number standard series were made by cloning the target ampliconinto a pCR2.1 plasmid (Invitrogen, Carlsbad, Calif.), and making adilution series, prepared in dilution buffer (10 mM Tris-HCl [pH 8.0],100 μg/mL yeast tRNA), for quantifying the copy number. The cocktail wasamplified using LightCycler® 480 Probes Master kit #04707494001 (RocheDiagnostics, USA). A 96-well microplate was demarcated and labeled, and8.0 μL of master mix was added per well. A sealing foil was gentlyattached to the microplate. The plate was centrifuged for 1 minute at3,000 rpm in a Qiagen microplate centrifuge. The sealing foil wasremoved, and 2.0 μL of thawed, diluted synthesized cDNA strands orplasmid DNA were added. A foil seal was firmly affixed to the plate andcentrifuged as previously described. A PCR program was run as follows:i) activate 95° C. for 10 minutes; ii) denature 95° C. for 10 sec (@4.8° C./sec); iii) anneal/extend 56° C. for 40 sec (@ 2.5° C./sec); iv)acquire 72° C. for 1 sec (@ 4.8° C./sec); steps ii-iv were repeated 45more times; vi) cool to 38° C. for 5 sec.

Selection of T₀ Plants for Excisor T₁ Production.

A total of 54 hygromycin-resistant plants were regenerated, and 34plants were found to have 1-2 transgene copies based on hydrolysis probeassay. Southern analysis identified 12 single-copy events of which 7displayed bands consistent with intact HPT and ZFN PTUs. T₀ pDAS5381events displaying single copy transgene, full length PTU, and expressingFokI, pDAS5381-18, pDAS5381-49 and pDAS5381-56, were self-pollination toproduce T₁ seed.

Example III: Generation and Selection of T₁ Plants

Selfing of T₀ Plants to Produce Homozygous T₁ Plants.

The following T₀ plant events: pDAS5380-3; pDAS5380-18; pDAS5380-46;pDAS5381-18; pDAS5381-49; and pDAS5381-56 were grown to maturity andself-fertilized to produce T₁ seed. Following germination, T₁ plantsthat were homozygous for the pDAS5380 and pDAS5381 constructs were usedfor transgene deletion. According to Mendelian inheritance, crossing thepDAS5381 homozygous single copy T₁ plants with the pDAS5380 homozygoussingle copy T₁ plants produce an F₁ population containing a heterozygoussingle copy of both the pDAS5381 and pDAS5380 constructs. The progeny ofthis cross was expected to contain one copy of the GUS reporter gene. Assuch, an F₁ plant not expressing GUS indicates that the GUS PTUexpression cassette has been excised.

T₀ plants were grown under a 16:8-hour photoperiod, with daytime andnighttime temperature between 22-24° C. When the primary flowering stembegan to elongate and form flower buds, the entire plant was coveredwith a selfing bag to prevent outcrossing. Seeds derived fromself-pollination were harvested about eight weeks after transplanting.The seed from the self-fertilized plants was collected and sewn intosoil. The resulting T₁ populations were grown in the greenhouse underthe conditions described above.

Molecular Screening of T₁ Plants.

Zygosity Assay.

An assay to quantify the zygosity of the T₁ plants was completed usingthe hydrolysis probe method described, supra (Copy Number Assay). Theanalysis of real time PCR data was performed and the number of transgenecopies contained in the T₁ plants was determined by comparison to a copynumber control. For this, a sample of genomic DNA from the parent T₀plant which was previously shown to contain a single copy calibrator wasincluded. Homozygous pDAS5380 and pDAS5381 T₁ plants were identified.

GUS Expression Assay.

It was important to identify expressing events for advancement to thecrossing experiments. The pDAS5380 T₁ plants were assayed using theprotocol described, supra (GUS Expression Assay). The pDAS5380 plantswhich were selected as homozygous for the pDAS5380 construct from thezygosity assay, supra, were tested. All of the plants stained blue.

GFP Expression Assay.

The pDAS5380 T₁ plants were assayed using the protocol described, supra(GFP Expression Assay). The pDAS5380 plants which were selected ashomozygous for the pDAS5380 construct from the zygosity assay, supra,were tested. All of the tested plants were positive for GFP expression.

FokI Expression Assay.

Quantitative Real-Time PCR (qRT-PCR) was used to quantify the mRNAexpression of the zinc finger nuclease in homozygous pDAS5381 T₁ tobaccoplants transformed with pDAS5381. The protocols described, supra (FokIExpression Assay), were used for the screening of T₁ plants to confirmthat the zinc finger nuclease was expressing, and to identify the eventswhich would produce robust quantities of zinc finger nuclease forexcising the GUS PTU expression cassette.

Selection of T₁ Plants.

T₁ pDAS5380 events were screened for zygosity and expression of GUS andGFP.

T₁ pDAS5381 events were screened for zygosity and expression of FokI.Based on these results, T₁ events were selected for crossing. Theseevents were identified as optimal, as they were homozygous, single copy,full length, transgene-expressing events. In addition, sibling-nullpDAS5381 plants were retained for use as controls. These events do notcontain the zinc finger nuclease PTU expression cassette. The transgenewas not inherited by these T₁ plants as a result of transgenesegregation. The selected events were grown to maturity and crossed toproduce F₁ plants to test transgene excision via the zinc fingernuclease. The crossing strategy is set forth below.

Crossing of the Homozygous T₁ Plants for Producing an F₁.

Selected pDAS5380 plants were crossed with select pDAS5381 plants. Inaddition, reciprocal crosses were made so that parents were both maleand female (Table 2). The plants were crossed by hand; pollen from theanthers of a mature male parent was introduced to the stigma of themature female parent. Plants ready for crossing were removed from theother plants to reduce the likelihood that unintended pollen wouldfertilize the female tobacco plants. Female plants were emasculated(anthers removed prior to dehiscence) using forceps ˜15-30 minutes priorto being pollinated by the male flower. Flowers were selected foremasculation by observing the anthers and the flower color. Newly openedflowers were bright pink around the edges and the anthers were stillclosed. Flowers containing anthers which were opened or partially openedwere not used. Multiple flowers from a stem of the tobacco plant wereemasculated and fertilized. The additional flowers on the stem (e.g.,already fertilized pods, old flowers, very young buds, etc.) wereremoved with forceps to ensure that the only pods to form on the branchwere from controlled crosses. The branch was labeled with a pollinationtag listing the cross made, how many crosses were made, and thepollination date. The anthers from the male parent were totally removedfrom the male plant using forceps, and used to fertilize the emasculatedfemale. The dehiscing male anthers were rubbed onto the sticky receptivefemale stigma until the stigma was coated with pollen. The stigma wascoated several times to reduce the chance of any unintended pollenhaving access to pollinate the female stigma. The seed from thefertilized plants was collected and sewn into soil. The resulting F₂progeny plants were grown in the greenhouse under the conditionsdescribed above.

TABLE 2 Crossing experiment matrix. Target Excisor Events Events 5381-185381-49 5381-56 5380-03 5381-18-17 × 5381-49-16 × 5381-56-5 × 5380-3-65380-3-12 5380-3-21 5380-18 5381-18-22 × 5381-49-16 × 5381-56-37 ×5380-18-17 5380-18-22 5380-18-22 5380-46 5381-18-17 × 5381-49-10 ×5381-56-5 × 5380-46-15 5380-46-15 5380-46-15 Null 5381-56-12 ×5381-56-12 × 5381-56-12 × Events 5380-3-10 and 5380-3-10 and 5380-3-10and 5380-25-10 5380-25-10 5380-25-10

Example IV: Analysis of F₁ Plants for ZFN-Mediated Transgene Deletion

GUS Assay.

The F₁ plants were tested for GUS expression by histochemically stainingleaf material. The GUS screen was a preliminary test to identify eventswhich had undergone ZFN—mediated transgene deletion. The results of theGUS screen were not intended to be conclusive, but rather an indicatorto identify plants for further molecular analysis. The F₁ plants wereassayed using the protocol described, supra (GUS Expression Assay). Theresults are listed in Table 3.

TABLE 3 GUS expression in F1 hybrids. Target Events 5380-03 5380-185380-46 Excisor Reciprocal Plants Plants Plants Events Cross AssayedGUS- % Assayed GUS- % Assayed GUS- % 5381-18 ♀ 479 15 3.1 490 3 0.6 4507 1.6 ♂ 459 44 9.6 480 21 4.4 — — — 5381-49 ♀ — — — 452 157 34.7 474 326.8 ♂ 465 67 14.4 467 17 3.6 485 69 14.2 5381-56 ♀ 437 4 0.9 476 0 0 4653 0.7 ♂ — — — 470 7 1.5 450 3 0.7 NULL ♀ — — — 441 8 1.8 453 11 2.4 ♂ —— — 446 4 0.9 490 11 2.2

Southern Blot Analysis.

Southern blot analysis was used to provide molecular characterization ofthe excision of the GUS PTU expression cassette by the zinc fingernuclease. This data demonstrated the excision of the GUS PTU expressioncassette in a sub-set of events, the non-excision of the GUS PTUexpression cassette in another sub-set of events, and a sub-set ofchimeric events which contained both excised and non-excised GUS PTUexpression cassette. Detailed Southern blot analysis was conducted usinga probe specific to the GFP PTU expression cassette. The hybridizationof the probe with genomic DNA that had been digested with specificrestriction enzymes identified DNA fragments of specific molecularweights. These patterns could be analyzed to identify events thatcontained an excised GUS PTU expression cassette, contained an intactGUS PTU expression cassette, or were chimeric and contained both theexcised and intact GUS PTU expression cassette.

A restriction digest was completed for 10 μg of each sample in 1× Buffer4 and 100 Units of NdeI (New England BioLabs, Ipswich, Mass.) in a finalvolume of 350 μL for a 10-fold over-digestion. Samples were incubated at37° C. overnight. The digested DNA was concentrated by re-precipitationwith Quick Precipitation Solution™ (Edge Biosystems, Gaithersburg, Md.)according to the manufacturer's suggested protocol. Recovered digest wasresuspended in 30 μL of 1× loading buffer and incubated at 65° C. for 30minutes. Resuspended samples were loaded onto a 0.8% agarose gelprepared in 1×TAE (0.8M Tris-acetate [pH 8.0]/0.04 mM EDTA) andelectrophoresed overnight at 1.1 V/cm in 1×TAE buffer. The gel wassequentially subjected to denaturation (0.2 M NaOH/0.6 M NaCl) for 30minutes, and neutralization (0.5 M Tris-HCl [pH 7.5]/1.5 M NaCl) for 30minutes. Transfer of DNA fragments was performed by passively wicking20×SSC solution overnight through the gel onto treated Immobilon™NY+(Millipore, Billerica, Mass.) by using a chromatography paper wickand paper towels. Following transfer, the membrane was briefly washedwith 2×SSC, cross-linked with the Stratalinker™ 1800 (Stratagene, LaJolla, Calif.), and vacuum baked at 80° C. for 3 hours. Blots wereincubated with prehybridization solution for 1 hour at 65° C. in glassroller bottles using a hybridization incubator. Probe was prepared fromPCR fragment containing the gfp coding sequence that was purified usinga Qiagen gel extraction kit and labeled with 50 μCi of α³²P-dCTP using alabeling kit. Blots were hybridized overnight at 65° C. with denaturedprobe added directly to hybridization buffer to approximately 2 millioncounts per blot per mL. Following hybridization, blots were sequentiallywashed at 65° C. with 0.1×SSC/0.1% SDS for 40 minutes. Blots wereexposed using phosphor imager screen and imaged using a MolecularDynamics Storm860™ imaging system. The results of the blots are shown inFIG. 5.

Plant Transcription Unit PCR Analysis.

PCR reactions were performed to characterize the excision of the GUS PTUexpression cassette. Primers were designed which bound to the MARsequence and the ORF 23 3′ UTR sequence (the 3′ UTR for the GFP PTUexpression cassette). This PCR amplicon spans the GUS PTU expressioncassette region which is expected to be excised. As such, the use ofthese PCR primers can detect events in which the GUS PTU expressioncassette was excised, events in which no excision occurred, and chimericevents in which the GUS PTU expression cassette was not uniformlyremoved within the event. Amplification of a 6.7 kb fragment indicatesthat there is no excision, whereas amplification of a 2.4 kb fragmentsuggests that the GUS PTU expression cassette had been excised.Amplicons containing fragments of both sizes indicate that the GUS PTUexpression cassette was not completely removed.

Genomic DNA was isolated from tobacco leaf tissue using the DNeasy™Plant Maxi kit, and quantified using the Quant-IT™ Pico Green DNA assaykit as described, supra. Plant Transcription Unit PCR (PTU PCR) wasperformed using a Tetrad2™ thermocycler (BioRad, Hercules, Calif.).Oligonucleotide primers were designed to amplify the PTU usingVectorNTI™ Software. For amplification, Ex Taq Polymerase™ (TaKara,Otsu, Shiga, Japan) was prepared at 1× final concentration in a 25 μLvolume singleplex reaction containing 1.2 μM of each primer (SEQ IDNOs:16 and 17), 0.2 mM dNTP, 2% DMSO, 1.25 units of TAQ using 4 ng ofgDNA template. A three-step amplification reaction was performed asfollows; 3 minute initial denaturation at 94° C. and 33 cycles of 30seconds of 94° C., 6 minutes of 65.5° C., 30 seconds of 72° C., with afinal extension at 72° C. for 10 minutes. An aliquot of the PCR productwas run on a 1% gel with ethidium bromide using a 1 Kb+ marker(Invitrogen, Carlsbad, Calif.) to determine product size. Results of thePTU PCR reactions are shown in FIG. 6.

Sequencing of PTU PCR Products.

The 2.4 kb bands from the PTU PCR reactions were excised from the geland DNA was purified using the Qiagen Qiaex II™ gel extraction kit(Qiagen, Germantown, Md.). The purified fragments were ligated into thepCR2.1 TOPO-TA™ cloning vector (Invitrogen, Carlsbad, Calif.). Presenceof a cloned PCR amplicon within the pCR2.1 vector was confirmed viarestriction enzyme digestion. Clones containing the amplified bands weresequenced. The sequences of the junction resulting from the removal ofthe GUS PTU expression cassette are listed in FIGS. 7a and 7b . Theentire PTU expression cassette was removed. The only sequences remainingare rearranged zinc finger binding sites which flanked the GUS PTUexpression cassette. In addition, several PCR amplicons containeddeletions which extended into the Actin 2 promoter of the GFP PTUexpression cassette.

Restriction Enzyme Analysis of 6.7 kb Band.

The PCR amplicons of the larger 6.7 kb band were analyzed viarestriction enzyme digestion. These fragments were digested with EcoRI,and with NcoI/SacI restriction enzymes (New England Biolabs, Ipswich,Mass.). The sizes of the resulting bands were analyzed to confirm thatthe amplified fragments spanned the non-excised pDAS5380 transgenegenomic insert.

Self-Fertilization of F₁ Plants to Produce F₂ Progenies.

A representative group of the F₁ plants described above wereself-fertilized to produce F₂ progenies. Table 5 lists the plants thatwere selected and their F₁ phenotype and genotype. Selected F₁ plantswere grown in a greenhouse under a 16:8-hour photoperiod, with daytimeand nighttime temperature between 22-24° C. When the primary floweringstem began to elongate and form flower buds, the entire plant wascovered with a selfing bag to prevent out-crossing. Seeds derived fromself-pollination were harvested about eight weeks after transplanting.The seed from the self-fertilized plants was collected and sewn intosoil. The resulting F₂ populations were grown in the greenhouse underthe conditions described above. The F₂ plants were analyzed for furthertransgene deletion and heritability of the deletion which had beencharacterized within F₁ plants.

Example V: Generation and Selection of T₁ Plants

Analysis of F₂ Progenies for Transgene and Heritability of Deletion.

GUS Analysis.

The F₂ plants were tested for GUS expression by histochemical stainingof leaf material. The plants were assayed using the protocol described,supra (GUS Expression Assay). The results are listed in Table 5. The GUSexpression data from the F₂ plants were as expected. The F₁ plants thatwere identified as containing an excised GUS PTU expression cassetteproduced F₂ plants that were 100% GUS negative, as confirmed byhistochemical staining. The absence of the GUS expression within theseF₂ plants confirms the F₁ data, which suggests that the GUS PTUexpression cassette was excised via zinc finger nuclease-mediatedtransgene deletion. Moreover, this data exemplifies the heritability ofthe deleted transgene into a subsequent generation.

The sibling-null control plants expressed GUS in about 75% of the F₂generation. The remaining plants (about 25%) in which GUS was notdetected via histochemical staining were expected. The GUS PTUexpression cassette is expected to segregate within the F₂ population atthe expected 3:1 ratio. The chimeric events which contained both excisedand non-excised GUS PTU expression cassettes in the F₁ segregated withinthe F₂. The majority of the plants expressed GUS.

TABLE 5 GUS expression in the F2 progenies. F1 Molecular/ # PhenotypicPlants Cross Cross Character- As- # # # Identity ization sayed GUS+ GUS−95 5380-46-1-15 × Excised/ 405 0 405 5381-49-1-10-003 GUS− 3075381-49-1-10 × Excised/ 480 0 480 5380-46-1-15.018 GUS− 180 5380-3-1-6 ×Excised/ 445 0 445 5381-18-1-17.002 GUS− 83 5380-46-1-15 × Excised/ 3750 375 5381-49-1-10-015 GUS− 77 5380-46-1-15 × Excised/ 427 0 4275381-49-1-10-021 GUS− 310 5381-49-1-10 × Excised/ 442 0 4425380-46-1-15.015 GUS− 265 5381-49-1-16 × Excised/ 471 0 4715380-3-1-12.017 GUS− 93 5380-46-1-15 × Excised/ 386 0 3865381-49-1-10-005 GUS− 4 5380-18-1-22 × Intact/GUS+ 473 356 1175381-49-1- (null) 14(null).017 214 5381-56-1-6(null) × Intact/GUS+ 480377 102 5380-46-1-23.010 (null) 292 5381-49-1-14(null) × Intact/GUS+ 481370 111 5380-18-1-22.013 (null) 189 5380-46-1-15 × Intact/ 456 345 1115381-56-1-5.001 GUS+ 335 5381-18-1-17 × Chimeric/ 449 326 1235380-46-1-15.011 GUS+ 350 5381-18-1-17 × Chimeric/ 457 342 1145380-18-1-22.020 GUS+ 331 5381-18-1-17 × Chimeric/ 452 347 1045380-46-1-15.016 GUS+ 53 5380-46-1-15 × Chimeric/ 470 359 1095381-18-1-17.014 GUS+

Green Fluorescent Protein ELISA Analysis.

Selected F₂ plants were tested for GFP expression by ELISA using theprotocol described, supra (GFP Expression Assay). GFP expression datafrom the F₂ plants were as expected. The F₁ plants expressed GFP inabout 75% of the F₂ generation. The remaining plants (about 25%) inwhich GFP was not detected via ELISA was expected. The GFP PTUexpression cassette is expected to segregate within the F₂ population atthe expected 3:1 ratio. The chimeric events which contained both excisedand non-excised GFP PTU expression cassettes in the F₁ segregated withinthe F₂. The majority of the plants expressed GFP.

PCR, Southern Blot, and GFP Analysis of F₂ Progenies.

Sixteen plants from three of the crosses listed in Table 5 (representingexcised, intact, and chimeric progenies) were kept for further molecularanalysis. These sixteen plants consisted of eight plants that were GUSpositive and eight plants that were GUS negative for the sibling nullcontrol and chimeric plants. The protocols described, supra (SouthernBlot Analysis; and Plant Transcription Unit PCR Analysis), were repeatedwith genomic DNA from the F₂ plants. Selected F₂ plants were tested forGFP expression by ELISA using the protocol described, supra (GFPExpression Assay). The molecular data confirm that plants which did notexpress GUS do not contain the intact GUS PTU expression cassette. GFPexpression segregated as expected. The results are summarized in FIGS.8-13.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein. The references discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention.

What is claimed is:
 1. A method for segregating a first polynucleotidefragment from a second polynucleotide fragment within a genome of aplant cell, wherein the first polynucleotide fragment and the secondpolynucleotide fragment are located in close proximity on a singlechromosome, the method comprising the steps of: (a) providing the plantcell comprising the first polynucleotide fragment and the secondpolynucleotide fragment located in close proximity on the singlechromosome, the plant cell further comprising at least a secondchromosome, wherein the first and second chromosomes are homologous orhomeologous chromosomes of each other; (b) introducing a site specificnuclease into the genome of the plant cell; (c) producing a doublestrand break in the first chromosome, wherein the double strand break inthe first chromosome is introduced between the first polynucleotidefragment and the second polynucleotide fragment; (d) producing a doublestrand break in the second chromosome, wherein the double strand breakin the second chromosome is introduced between the first polynucleotidefragment and the second polynucleotide fragment; and (e) obtaining aprogeny plant comprising a modified genome, wherein the firstpolynucleotide fragment and the second polynucleotide fragment segregatefrom one another.
 2. The method of claim 1, wherein the site specificnuclease comprises a zinc finger nuclease, a TALEN nuclease, aCRISPR-Cas9 nuclease, a meganuclease, or a leucine zipper nuclease. 3.The method of claim 1, wherein the site specific nuclease is deliveredto the plant cell by intra-genomic recombination or via direct delivery.4. A plant produced by the method of claim
 1. 5. The plant of claim 4,wherein the plant is a transgenic plant.
 6. A plant part, fruit or seedobtained from the plant of claim
 4. 7. A method to segregate a firstpolynucleotide fragment from a second polynucleotide fragment within thegenome of a progeny plant, the method comprising the steps of: (a)providing a plant cell comprising the first polynucleotide fragment andthe second polynucleotide fragment located in close proximity on asingle chromosome, the plant cell further comprising at least a secondchromosome, wherein the chromosomes are homologous or homeologouschromosomes of each other; (b) creating a double strand break in thefirst and second chromosome, wherein the double strand break in thehomologous or homeologous chromosomes is introduced between the firstpolynucleotide fragment and the second polynucleotide fragment; (c)segregating the first polynucleotide fragment from the secondpolynucleotide fragment located in close proximity on the singlechromosome of the plant cell; and (d) producing a progeny plant thatdoes not contain the first polynucleotide fragment within the genome ofthe progeny plant.
 8. The method of claim 7, wherein the double strandbreak is produced by a site specific nuclease.
 9. The method of claim 8,wherein the site specific nuclease comprises a zinc finger nuclease, aTALEN nuclease, a CRISPR-Cas9 nuclease, a meganuclease, or a leucinezipper nuclease.
 10. The method of claim 8, wherein the site specificnuclease is delivered to the plant cell by intra-genomic recombinationor via direct delivery.
 11. A plant produced by the method of claim 7.12. The plant of claim 11, wherein the plant is a transgenic plant. 13.A plant part, fruit or seed obtained from the plant of claim
 11. 14. Amethod for segregating the inheritance of a first transgene from asecond transgene in a progeny plant, the method comprising the steps of:(a) crossing a first parent plant with a second parent plant; (b)producing a double strand break between the first transgene and thesecond transgene; and (c) obtaining the progeny plant.
 15. The method ofclaim 14, further comprising screening the progeny plant for segregationof the first transgene from the second transgene in the progeny plant.16. The method of claim 14, wherein the double strand break is producedby a site specific nuclease.
 17. The method of claim 16, wherein thesite specific nuclease comprises a zinc finger nuclease, a TALENnuclease, a CRISPR-Cas9 nuclease, a meganuclease, or a leucine zippernuclease.
 18. The method of claim 16, wherein the site specific nucleaseis delivered to the plant by intra-genomic recombination or via directdelivery.
 19. A plant produced by the method of claim
 14. 20. The plantof claim 19, wherein the plant is a transgenic plant.
 21. A plant part,fruit or seed obtained from the plant of claim 19.